Use of des-aspartate-angiotensin I

ABSTRACT

The use of des-aspartate-angiotensin I, its derivatives and/or analogue thereof in medicine is described. In particular, a method for the treatment and/or prophylaxis of viral infections, for inducing hypoglycaemia and/or for reducing hyperglycaemia, and/or for treatment of hypoglycaemia-related conditions is described.

RELATED APPLICATIONS

The present application is a U.S. National Phase Application ofInternational Application PCT/SG2006/000264 (filed: Sep. 8, 2006) whichclaims the benefit of U.S. Provisional Application No. 60/715,156 (filedSep. 9, 2005), both of which are herein incorporated by reference intheir entirety for all purposes.

FIELD OF THE INVENTION

The present invention relates to the use of des-aspartate-angiotensin I(des-asp-ang I), its derivative, a functional part and/or analoguethereof in medicine.

BACKGROUND OF THE INVENTION

Viral respiratory tract infection is the most common illness in humansand the economic cost for non-influenza-related viral respiratory tractinfection approaches $40 billion in the US (Fendrick A M et al., 2003),while influenza causes tens of millions of day of restricted activity,bed disability and work loss (Sullivan K M et al, 1993). There is alsothe ever present threat of a pandemic flu outbreak and the apparentunstoppable AIDS virus spreading far and wide. With the ability ofviruses to mutate by genetic drift and genetic shift, new viruses thatare not susceptible to existing vaccines will continue to come intobeing. There is, therefore, a need to develop more effective antiviraldrugs.

Another medical condition, prolonged or chronic hyperglycemia, whereinblood glucose levels are elevated from long periods, produces manydetrimental effects. This condition is exemplified by diabetes. Diabetesexacts a huge toll in money and human suffering, accounting for morethan 100 billion dollars of healthcare costs annually in the UnitedStates (DiRamond J, 2003). The number of cases worldwide is estimated at150 million with an equal number of undiagnosed cases in the First Worldcountries and eight times more undiagnosed cases in the Third World(Levitt N S et al, 1993). The disease is characterised by high bloodsugar (glucose) resulting from the defects in either insulin secretionor decreased sensitivity of the body's cells to the action of insulin,leading to a condition of hyperglycemia. Type I diabetes is currentlytreated by preprandial administration of exogenous insulin and dietaryrestriction. Current therapy of type 2 diabetes includes lifestylemodifications and the use of a variety of pharmacological agents thataim to increase insulin secretion, decrease hepatic glucose production,and/or increase insulin actions. Despite these approaches, good glycemiccontrol is beyond the reach of most diabetic individuals, and the stateof prolonged or chronic hyperglycemia can cause cardiovascular diseases,stroke, blindness, kidney failure, neurological dysfunction, necrosisand gangrene of extremities. The search for better and more specificagents with physiological properties for hyperglycemia is highlywarranted.

Accordingly, any new and improved treatment of viral infections and/orconditions relating or due to hyperglycemia will be welcome.

The compound des-aspartate-angiotensin I is an endogenous angiotensinpeptide (Sim M k et al, 1994, a,b). It is formed or derived fromangiotensin I by a specific aminopeptidase present in blood vessels andthe hypothalamus (Sim M K, Qui X S, 1994). Previous studies suggestedthe use of des-aspartate-angiotensin I in cardiovascular and renalactions (Sim U.S. Pat. Nos. 5,773,415, 6,100,237, 6,589,938B2 andUS2003/0086920). However, no further uses are taught or suggested fordes-aspartate-angiotensin I.

SUMMARY OF THE INVENTION

The present invention addresses the problems above, and in particular,provides new and effective means of treatment and/or prophylaxis ofviral infections and hyperglycemia-related conditions. In particular,the present inventor has surprisingly found that at least one derivativeof angiotensin I may be used in inducing hypoglycemia and/or forreducing hyperglycemia and/or for the treatment of hyperglycemia-relatedconditions as well as in the treatment and/or prophylaxis of viralinfections. More in particular, the at least one derivative ofangiotensin I is des-aspartate-angiotensin I, its derivative, afunctional part, and/or an analogue thereof.

Accordingly, in one aspect, the present invention provides a method forthe treatment and/or prophylaxis of at least one viral infectioncomprising administering to a subject des-aspartate-angiotensin I, itsderivative, a functional part and/or an analogue thereof.

In another aspect, the present invention provides a method of inducinghypoglycaemia and/or reducing hyperglycaemia in a subject, the methodcomprising administering to the subject des-aspartate-angiotensin I, itsderivative, a functional part and/or analogue thereof.

There is also provided a method of treatment and/or prophylaxis of atleast one hyperglycaemia-related condition comprising administering to asubject des-aspartate-angiotensin I, its derivative, a functional partand/or analogue thereof, provided the at least onehyperglycaemia-related condition is not a renal-related disorder.

There is also provided des-aspartate-angiotensin I, its derivative, afunctional part and/or analogue thereof, and/or a pharmaceuticalcomposition thereof, for use in the treatment and/or prophylaxis of atleast one viral infection.

There is also provided des-aspartate-angiotensin I, its derivative, afunctional part and/or analogue thereof, and/or a pharmaceuticalcomposition thereof, for use in the induction of hypoglycaemia and/orreduction of hyperglycaemia. There is also provideddes-aspartate-angiotensin I, its derivative, a functional part and/oranalogue thereof, and/or a pharmaceutical composition thereof, for usein the treatment and/or prophylaxis of at least onehyperglycaemia-related condition, provided the at least onehyperglycaemia-related condition is not a renal-related disorder.

There is also provided the use of des-aspartate-angiotensin I, itsderivative, a functional part and/or analogue thereof for thepreparation of a medicament for the treatment and/or prophylaxis of atleast one viral infection; for the induction and/or reduction ofhyperglycaemia; as well as for the treatment and/or prophylaxis of atleast one hyperglycaemia-related condition, provided the at least onehyperglycaemia-related condition is not a renal-related disorder.

In another aspect, the present invention provides a kit for thetreatment and/or prophylaxis of at least one viral infection comprisingdes-aspartate-angiotensin I, its derivative, a functional part and/oranalogue thereof.

In another aspect, the present invention provides a kit for theinduction of hypoglycaemia and/or reduction of hyperglycaemia comprisingdes-aspartate-angiotensin I, its derivative, a functional part and/oranalogue thereof. There is also provided a kit for the treatment and/orprophylaxis of at least one hyperglycaemia-related condition providedthe at least one hyperglycaemia-related condition is not a renal-relateddisorder, the kit comprising des-aspartate-angiotensin I, itsderivative, a functional part and/or analogue thereof.

Under the relevant aspects above, the at least one viral infection maybe a respiratory viral infection. In particular, the viral infection maybe an influenza virus infection such as an influenza A infection.

The at least one hyperglycaemia-related condition may be type Idiabetes, obesity, and/or bulimia nervosa.

The des-aspartate-angiotensin I, its derivative, a functional partand/or analogue thereof, may be administered in a pharmaceuticallyand/or therapeutically effective amount. The des-aspartate-angiotensinI, its derivative, a functional part and/or analogue thereof, maycomprise at least one pharmaceutically-acceptable carrier, excipient,diluent and/or adjuvant.

The kits may further comprise information and/or instructions pertainingto their use.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

Figures:

FIG. 1 shows the effect of des-aspartate-angiotensin I on weight lossand survival of influenza A virus-infected female BALB/c mice. Six to 7week-old mice were intranasally infected with 50 μL passage-6 lunghomogenate (equivalent to 2.5×105 TCID50 of influenza A in MDCK cells).Group of 5-9 mice were then orally administered with one of thefollowing doses of des-aspartate-angiotensin I:—300 nmole/kg/dat for 8days (n=5), 600 nmole/kg/day for 8 days (n=9), and 120 nmole/kgadministered on Day 2 post-infection (n=9, only one dose wasadministered). The control group (n=8) was similarly administered waterfor 8 days. The weight and survival of the mice were recorded daily for16 days.

FIGS. 2 to 6 show the effect of des-aspartate-angiotensin I on bloodglucose profile animal models of diabetes.

FIG. 2 shows the effect of des-aspartate-angiotensin I on blood glucoseprofile in diabetic KK-Ay mice. The diabteic KKAy mice were divided intofour groups consisting of 6 animals per group. Animals in the controlgroup were intraperitoneally administered 0.1 mL saline. Animals in thesecond, third, and fourth groups were similarly administered with 100,200, 400 nmole/kg des-aspartate-angiotensin I, respectively. Treatmentwith saline and des-aspartate-angiotensin I was carried out daily for 4weeks. Following this, animals were fasted overnight for 16 hours andoral glucose tolerance test was performed as follows: blood waswithdrawn from the orbital sinus for blood glucose determination (timeof withdrawal was designated as 0 time), animals were then orallyadministered glucose (2 g/kg), and blood was withdrawn at 30, 60 and 120min for blood glucose determination. The blood was allowed to clot andblood glucose was measured as serum glucose using a commercial glucosekit from Thermo Electron Corporation, Australia. *Significantlydifferent from the corresponding values of the control untreated mice(p<0.05, ANOVA followed by post hoc Tukey test).

FIG. 3 shows the effect of orally administered des-aspartate-angiotensinII on blood glucose profile in diabetic GK rats at 4 and 6 weeks oftreatment. The experiment was similarly carried as for KKAy mice.Control animals were administered (by gavage) 0.2 mL of water and groups1 and 2 animals were similarly administered 400 and 600 nmole/kgdes-aspartate-angiotensin I in 0.2 mL water, respectively, for a period8 weeks. Oral glucose tolerance test (determined at 30 min) was carriedout after 4 and 6 weeks of treatment. N=5 to 6. *Significantly differentfrom the corresponding values of the control untreated rat (p<0.05,ANOVA followed by post hoc Tukey test). Data obtained after 8 week oftreatment are given in another figure, FIG. 4. Note: The data show thatorally administered DAA-1 exerts hypoglycaemic action in diabetic GKrats after 6 weeks of treatment.

FIG. 4 shows the effect of orally administered des-aspartate-angiotensinI on blood glucose profile in diabetic GK rats at 8 weeks of treatment.This figure gives the data of a full oral glucose tolerance testconducted at 8 weeks of the same experiment described in FIG. 3.*Significantly different from the value of the corresponding control(p<0.05, ANOVA post hoc Tukey test).

FIG. 5 shows the Effects of des-aspartate-angiotesnin I oninsulin-induced translocation of GLUT-4 in skeletal muscle of diabeticGK rats. Upper panel: Representative Western blot of plasma membraneGLUT4 in skeletal muscle of des-aspartate-angiotensin I treated and nontreated diabetic GK rats. Lower panel: Relative (to the non insulinstimulated samples) levels of plasma membrane GLUT4 in skeletal muscleof des-aspartate-angiotensin I treated and control diabetic GK rats. N=3for each histogram. *Significantly different from the value of thecorresponding control (p<0.05, ANOVA post hoc Tukey test).

FIG. 6 shows the effect of des-aspartate-angiotesnin I on serum insulinlevel of diabetic GK rats. Serum from 8 weeks treated animals (asdescribed in EXAMPLE 9) were assayed for insulin concentration by the“Ultra Senstive Rat Insulin ELISA kit” (Crystal Chem. Inc., IL, USA).The values were from serum taken at 30 min of the oral glucose tolerancetest. There were no significant difference between the values of thecontrol and treated group.

TABLES

-   Table 1 shows some examples of unnatural amino acids contemplated by    the present invention.-   Tables 2 and 3 show the effect of des-aspartate-angiotensin I in    animal models of influenza.-   Table 2 shows the percentage weight change and survival of influenza    A virus-infected BALB/c mice treated with orally-administered    des-aspartate-angiotensin I-   Table 3 shows the percentage weight change and survival of influenza    A virus-infected BALB/c mice treated with    intraperitoneally-administered des-aspartate-angiotensin I-   Tables 4 and 5 show the effects of des-aspartate-angiotensin I in    control of blood glucose levels in animal models of diabetes.-   Table 4 shows the data of the oral glucose tolerance test carried    out in diabetic KKAy mice that were treated with    intraperitoneally-administered des-aspartate-angiotensin I-   Table 5 shows data of oral the glucose tolerance test carried out in    diabetic KKAy mice that were treated with orally-administered    des-aspartate-angiotensin I.

DETAILED DESCRIPTION OF THE INVENTION

Bibliographic references mentioned in the present specification are forconvenience listed in the form of a list of references and added at theend of the examples. The whole content of such bibliographic referencesis herein incorporated by reference.

Des-aspartate angiotensin I have been previously described for use intreatment of certain disorders. For example, the effect of des-aspartateangiotensin I in segmental glomerulosclerosis rat model resembling renallesions in humans arising from various disorders including those arisingfrom diabetus mellitus was previously determined (US2003/0086920).

However, the prior art does not teach or suggest use ofdes-aspartate-angiotensin I, its derivative, a functional part and/oranalogue thereof for the treatment and/or prophylaxis of viralinfections and/or for the induction of hypoglycaemia and/or reduction ofhyperglycemia-related conditions. In particular, the present inventionrelates to the use of des-asparatate-angiotensin I in the treatment ofhyperglicemia-related condition(s), excluding renal-related disorders.In particular, the condition(s) treated according to the presentinvention do not include renal-related disorders described inUS2003/0086920.

The present invention relates to the use of des-aspartate-angiotensin I,its derivative, a functional part and/or analogue thereof for thetreatment and/or prophylaxis of viral infections and/or for theinduction of hypoglycaemia and/or the reduction of hyperglycaemia. Inparticular, the present invention relates to the use ofdes-aspartate-angiotensin I, its derivative, a functional part and/oranalogue thereof for the treatment and/or prophylaxis of influenza Ainfection and hyperglycaemia-related conditions as type I diabetes,obesity, and/or bulimia nervosa.

While researching the effects of des-aspartate-angiotensin I on thecardiopulmonary functions of influenza A virus-infected mice, it wassurprisingly discovered that des-aspartate-angiotensin I increased thesurvival of the animals.

Similarly, it was surprisingly discovered that des-aspartate-angiotensinI exerts marked hypoglycaemic effects in animal models ofhyperglycaemia. Accordingly, the present invention provides newtreatments, prophylaxes and/or pharmaceutical compositions for viralinfections and/or hyperglycemia-related conditions using itsdes-aspartate-angiotensin I, its derivative, functional part, and/oranalogue thereof.

Treatment of Viral Infections

The present invention provides a method for the treatment and/orprophylaxis of at least one viral infection and its symptoms comprisingadministering to a subject des-aspartate-angiotensin I, its derivative,a functional part and/or an analogue thereof. In particular, thedes-aspartate-angiotensin I may be administered in a therapeuticallyand/or pharmaceutically effective amount. More in particular, the methodmay comprise administering to a subject an therapeutically and/orpharmaceutically effective amount of at least one derivative ofangiotensin I, according to the present invention, for a time and underconditions sufficient for the onset of the viral infection and/or itssymptoms to be prevented, inhibited and/or delayed or the symptoms ofthe viral infection to be ameliorated. The derivative may bedes-aspartate-angiotensin I, or a derivative, homologue and/or analoguethereof.

The at least one viral infection may be a respiratory viral infection.In particular, the viral infection may be an influenza virus infectionsuch as an influenza A infection.

Induction of Hypoglycaemia/Reduction of Hyperglycemia

In another aspect, the present invention also provides a method ofinducing hypoglycaemia and/or reducing hyperglycemia in a subject, themethod comprising administering to the subject des-aspartate-angiotensinI, its derivative, a functional part and/or analogue thereof.

In particular, the present invention provides a method of treatmentand/or prophylaxis of at least one hyperglycaemia-related conditioncomprising administering to a subject des-aspartate-angiotensin I, itsderivative, a functional part and/or analogue thereof, wherein the atleast one hyperglycaemia-related condition excludes renal-relateddisorders.

In particular, the present invention provides a method for the treatmentand/or prophylaxis of at least one of the following states orconditions: hyperglycaemia, particularly prolonged or uncontrolledhyperglycaemic conditions as in type I diabetes, obesity, bulimianervosa excluding renal disorders arising from hyperglycaemia as in Type2 diabetes (diabetes mellitus), the method comprising administering to asubject at least one derivative of angiotensin I. In particular, the atleast one derivative of angiotensin I is des-aspartate-angiotensin I,its derivative, a functional part and/or analogue thereof. Inparticular, the angiotensin I derivative is administered for a time andunder conditions sufficient for the condition and/or its symptoms to beprevented, inhibited, delayed and/or ameliorated.

The present invention also provides des-aspartate-angiotensin I or apharmaceutical composition therefore for use in the treatment and/orprophylaxis of hyperglcaemia, in particular prolonged or uncontrolledhyperglycaemic conditions as in type I diabetes, obesity, bulimianervosa excluding renal disorders arising from hyperglycemia as in Type2 diabetes mellitus, The pharmaceutical composition may comprise atleast one derivative of angiotensin I according to the present inventionand, optionally, a pharmaceutically acceptable carrier, excipient,adjuvant and/or diluent.

The compositions may be administered in a therapeutically and/orpharmaceutically effective dose, and/or for a time and under conditionssufficient for its symptoms to be prevented, inhibited or delayed and/orameliorated.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary to achieve the desired therapeutic result,such as to prevent, inhibit and/or delay the onsethyperglycaemia-related disorders and/or ameliorate the symptoms ofhyperglycaemia-related disorders. While the effective amount may varyaccording to various factors such as the disease state, age, sex, andweight of the individual.

A derivative of des-aspartate-angiotensin I includes any mutant,fragment, part and/or portion of angiotensin I ordes-aspartate-angiotensin I including molecules comprising single ormultiple amino acid substitutions, deletions and/or additions toangiotensin I or to des-aspartate-angiotensin I. The processes ofsubstitutions, deletions and/or additions may result in a derivative orpeptide that has more amino acids than the original angiotensin I ordes-aspartate-angiotensin I. The processes of substitutions, deletionsand/or additions may result in a derivative or peptide that has moreamino acids than the original angiotensin I or des-aspartate angiotensinI

The preferred derivative in accordance with the present invention isdes-aspartate-angiotensin I or a derivative, homologue and/or analoguethereof. Reference to a homologue and/or an analogue includes a mimotopeor peptide and/or analogue mimetic. Such derivatives, homologues and/oranalogues may function in place of des-aspartate-angiotensin I and/orits equivalent or they may act as an agonist thereof or, when necessary,an antagonist thereof.

As stated above, a derivative includes single or multiple amino acidsubstitutions, additions and/or deletions to des-aspartate-angiotensin Iand/or angiotensin I. The final derivative is a peptide having similarfunctions and consists of no less than two amino acid sequence of theoriginal angiotensin I or des-aspartate-angiotensin.

Amino acid insertional derivatives of des-Asp-angiotensin I includeamino and/or carboxyl terminal fusions as well as intra-sequenceinsertions of single or multiple amino acids. Insertional amino acidsequence variants are those in which one or more amino acid residues areintroduced into a predetermined site in the des-aspartate-angiotensin Ialthough random insertion is also possible with suitable screening ofthe resulting product.

Deletional variants are characterized by the removal of one or moreamino acids from the sequence. Substitutional amino acid variants arethose in which at least one residue in the sequence has been removed anda different residue inserted in its place.

Where the des-aspartate-angiotensin I is derivatized by amino acidsubstitution, the amino acids are generally replaced by other aminoacids having like properties, such as hydrophobicity, hydrophilicity,electronegativity, bulky side chains and the like. Amino acidsubstitutions are either of single or multiple residues. Amino acidinsertions will usually be in the order of about 1-7 amino acid residuesand deletions will range from about 1-7 residues. Preferably, deletionsor insertions are made in adjacent pairs, i.e. a deletion of tworesidues or insertion of two residues.

Homologues include functionally, structurally or stereochemicallysimilar polypeptides from, for example, other sources such as forlivestock animals, laboratory test animals or primates. The similarpeptides may also be homologues of des-aspartate-angiotensin I.

Analogues and mimetics include molecules which contain non-naturallyoccurring amino acids as well as molecules which do not contain aminoacids but nevertheless behave functionally the same as thedes-aspartate-angiotensin I. Analogues contemplated herein includemodifications to side chains, incorporation of unnatural amino acidsand/or their derivatives during peptide synthesis and the use ofcrosslinkers and other methods which impose conformational constraintson the peptide molecule or their analogues.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, but are not limited to, use of norleucine,4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid,6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine,ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid,2-thienyl alanine and/or D-isomers of amino acids.

Crosslinkers can be used, for example, to stabilize 3D conformations,using homo-bifunctional crosslinkers such as the bifunctional imidoesters having (CH.2)n spacer groups with n=1 to n=6, glutaraldehyde,N-hydroxysuccinimide esters and heterobifunctional reagents whichusually contain an amino reactive moiety such as N-hydroxysuccinimideand another group specific-reactive moiety.

All these types of modifications may be important to stabilize thesubject des-Asp-angiotensin I. This may be important if used, forexample, in the manufacture of a vaccine or therapeutic composition orin detection assays. Examples of unnatural amino acids contemplated bythe present invention are presented in Table 1.

TABLE 1 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmglncarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-α-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcylcopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycineNcoct D-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-α-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen α-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L--methylarginine MargL-α-methylasparagine Masn L--methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L--methylcysteine McysL-methylethylglycine Metg L--methylglutamine Mgln L-α-methylglutamateMglu L--methylhistidine Mhis L-α-methylhomophenylalanine MhpheL--methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL--methylleucine Mleu L-α-methyllysine Mlys L--methylmethionine MmetL-α-methylnorleucine Mnle L--methylnorvaline Mnva L-α-methylornithineMorn L--methylphenylalanine Mphe L-α-methylproline Mpro L--methylserineMser L-α-methylthreonine Mthr L--methyltryptophan MtrpL-α-methyltyrosine Mtyr L--methylvaline Mval L-N-methylhomophenylalanineNmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhecarbamylmethyl)glycine carbamylmethyl)glycine 1-carboxy-1-(2,2-diphenyl-Nmbc ethylamino)cyclopropaneHaving now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting tothe present invention.

EXAMPLES

Standard molecular biology techniques known in the art and notspecifically described were generally followed as described in Sambrookand Russel, Molecular Cloning: A Laboratory Manual, Cold Springs HarborLaboratory, New York (2001).

Example 1 Source of Materials

Des-aspartate-angiotensin was purchased from Bachem (Dubendorf,Switzerland). Des-aspartate-angiotensin I can be prepared by techniqueswell known in the art. Influenza A/Aichi/2/68 virus and Madin-DarbyCanine Kidney cells were purchased from ATCC. The virus has a titer of106.75 CEID50/0.2 ml in 2 days on II day old SPF CE. Six to 7 weeks oldfemale BALB/c mice were obtained from the Animal Center, NationalUniversity of Singapore. Six to 7 weeks old diabetic KKAy mice werepurchased from CLEA, Japan.

Example 2 Development of a Mouse Model for Influenza A Virus

Two BALB/c mice were intranasally infected with 50 μL of influenza Avirus. Two days later, the infected mice were sacrificed by cervicaldislocation and their lungs were removed. 0.3 g of lung tissue washomogenized in 1 mL of PBS containing 1000 units/L penicillin and 10μg/L streptomycin. The lung homogenate was cleared of connective tissueby centrifuging at 5,000 rpm for 5 minutes on a bench-top centrifuge.This homogenate was labeled as passage-1 lung homogenate. A second batchof 2 BALB/c mice was infected with 50 μL of passage-1 lung homogenate.The process of passaging was repeated and the virus virulence and titerin each passage of lung homogenate was monitored by assaying itsinfectivity in Madin-Darby Canine Kidney (MDCK) cells, which is a hostcell for influenza A virus. The virulence and titer of the influenza Avirus increased progressively with each passage. Fifty μL of passage-6lung homogenate, equivalent to 2.5×105 TCID50 of the virus in MDCKcells, were then used to infect 6-7 weeks old female BALB/c mice for thestudy of the antiviral action of des-aspartate-angiotensin I.

Example 3 Monitoring and Treatment (with Orally-AdministeredDes-aspartate-angiotensin I) of Influenza A Virus-Infected BALB/c Mice

BALB/c mice when infected with passage-6 lung homogenate lost weight andsuffered fatality. The following protocol described by Johasson et alwas used to study the effect of des-aspartate-angiotensin on theseparameters. Six to 7 weeks old female BALB/c mice were intranasallyinfected with 50 μL passage-6 lung homogenate. Groups of 5-9 mice wereorally administered with one of the following dose ofdes-aspartate-angiotensin I:—300 nmole/kg daily for 8 days, 600 nmole/kgdaily for 8 days, and 1200 nmole/kg on day 2 post virus infection (i.e.only one dose was administered). A group of 8 mice were similarlyadministered water for 8 days and served as the control group. Theweight and survival of the mice were recorded daily for 16 days.

Example 4 Effect of Orally-Administered Des-aspartate-angiotensin I onVirus-Infected BALB/c Mice

The results of the study are summarized in Table 2 and depicted inFIG. 1. The daily weight of each surviving animal was expressed as apercentage of its weight at Day 0 (i.e. day of virus inoculation). Thedata of the control and each treatment group were expressed as the meanI SEM. Significant differences were determined by one way ANOVA and posthoc Tukey test. The accepted level of significance was p<0.05.Des-aspartate-angiotensin I is an antiviral agent in term of its abilityto attenuate the weight loss and survival of the virus-infected animals.Effective antiviral activity was observed with a dose of 600 nmole/kgadministered daily for 8 days, and a single dose of 1200 nmole/kg givenon Day 2.

Example 5 Monitoring and Treatment (with Intraperitoneally-administeredDes-aspartate-angiotensin I) of Influenza A Virus-infected BALB/c Mice

Thirty BALB/c mice were infected with 55 μL passage-6 lung homogenateand the weight of each animal was monitored daily as described inEXAMPLES 3 and 4. Animals were intraperitoneally administered witheither 60 nmole/kg des-aspartate-angiotensin I in 0.1 ml of phosphatebuffer saline (PBS) or 0.1 ml PBS on the day when their weight loss was15% or greater during the first post-infection week. This treatment wascontinued for 7 days. Of the 30 animals, 14 animals lost more than 15%of their weight during the first post-infection week. Hence, 7 animalswere randomly chosen for des-aspartate-angiotensin I treatment and 7animals for vehicle treatment. The weight of the treated animals weremonitored till either the animal died or regained 100% of their initialweight. Surviving animals were euthanized and their lung homogenate wasprepared and assayed for influenza A virus as described in Example 2.

TABLE 2 Post Average Percentage Weight Change (weight of individualInfection animals at Day 0 was taken as 100%) Period 300 nmole/kg/ 600nmole/kg/ 1200 nmole/kg (in day day given as a single Days) Control (n =8) for 16 days (n = 5) for 16 days (n = 9) dose on Day 2 (n = 9) 0 100100 100 100 1 102.4 ± 2.2 102.1 ± 1.5  98.7 ± 1.3 103.5 ± 0.5 2  93.4 ±1.4  96.2 ± 2.0  95.8 ± 1.4  96.8 ± 0.6 3  88.7 ± 1.0  90.8 ± 2.4  92.0± 1.7  95.1 ± 0.8* 4  86.4 ± 1.3  85.8 ± 2.9  92.4 ± 1.8*  96.7 ± 1.2* 5 84.1 ± 2.3  83.2 ± 3.4  94.8 ± 1.8*  96.3 ± 2.1* 6  83.3 ± 3.6 94.1^(#) (3)  95.5 ± 2.1*  95.7 ± 3.1* 7  84.1 ± 4.6  95.7^(#)  97.8 ±2.6*  97.8 ± 4.0* 8  91.0 ± 5.4 (2)  97.9^(#)  96.7 ± 2.9  98.3 ± 4.3 9 91.8 ± 6.2  98.7^(#)  96.3 ± 3.5 101.1 ± 4.7 10  93.4 ± 7.1  98.1^(#) 95.6 ± 4.0 101.1 ± 4.8 11 101.6 ± 2.8 (1)  97.4^(#)  95.4 ± 4.5 105.9 ±1.3 (1) 12 102.4 ± 3.1  96.9^(#)  95.1 ± 5.0 106.1 ± 1.3 13  99.9 ± 5.8 96.8^(#)  95.5 ± 5.2 108.1 ± 1.2 14 103.0 ± 3.9  98.2^(#)  99.1 ± 3.4107.2 ± 1.3 15 105.4 ± 3.8  99.6^(#) 100.3 ± 4.2 109.4 ± 1.2 16 104.4 ±3.5 102.4^(#)  97.0 ± 4.8 108.4 ± 1.5 *Significantly different from thecorresponding control value (p < 0.05, ANOVA post hoc Tukey test) Boldnumbers within brackets indicate number of animals that died on the dayof recording. ^(#)Average value from two surviving mice i.e. no SEM.

Example 6 Effect of Intraperitoneally-administeredDes-aspartate-angiotensin I on Virus-infected BALB/c Mice

The data of the study are summarized in Table 3. The weight of eachanimal was expressed as a percentage of its weight at Day 0. All micethat were treated with saline died within a week. In thedes-aspartate-angiotensin I treated mice, only I mouse died. Six animalssurvived and regained their original weight. No influenza A virus wasdetected in the lung homogenate of the surviving mice. The results showthat des-aspartate-angiotensin I exerts effective antiviral action inpreventing influenza A virus-infected mice from dying.

Example 7 Effect of Intraperitoneally-AdministeredDes-aspartate-angiotensin I on Blood Glucose Profile in Diabetic KKAyMice

The diabetic KKAy mice were divided into four groups consisting of 6animals per group. Animals in the control group were intraperitoneallyadministered 0.1 mL saline. Animals in the second, third, and fourthgroups were similarly administered with 100, 200, 400 nmole/kgdes-aspartate-angiotensin I, respectively in 0.1 ml of PBS. Treatmentwith saline and des-aspartate-angiotensin I was carried out daily for 4weeks. Following this, animals were fasted overnight for 16 hours andoral glucose tolerance test was performed as follows: blood waswithdrawn from the orbital sinus for blood glucose determination (timeof withdrawal was designated as 0 time), animals were then orallyadministered glucose (2 g/kg), and blood was withdrawn at 30, 60 and 120min for blood glucose determination. The blood was allowed to clot andblood glucose was measured as serum glucose using a commercial glucosekit from Thermo Electron Corporation, Australia. Table 3 shows thatdes-aspartate-angiotensin I, administered at doses of 200 and 400nmole/kg/day for 4 weeks, significantly lowered the blood glucose levelat 30 and 60 min of the oral glucose tolerance test. The data,graphically displayed in FIG. 2, show that des-aspartate-angiotensin Ipossesses hypoglycaemic action and can be use to treat the symptoms ofhyperglycaemia.

Example 8 Dose-Range Finder Experiment for Orally AdministeredDes-aspartate-angiotensin I

A dose-range finder experiment to determine the hypoglycaemic dose fororally administered des-aspartate-angiotensin I was also carried out. Inthis experiment, the animals in each of the three treatment groups of 7diabetic KKAy mice were orally administered (by gavage) 200, 400, and600 nmole/kg/day des-aspartate-angiotensin I in volume of 0.1 mL,respectively. Animals in the control group were similarly administered0.1 mL water. The treatment period was 4 weeks. Oral glucose tolerancetest was carried out as described in Example 5, except that blood wassampled at 0 and 30 min. The data, presented in Table 4, show thatdes-aspartate-angiotensin I is an effective oral hyperglycemic agent atan oral dose of 600 nmole/kg.

Example 9 Effect of Orally-Administered Des-aspartate-angiotensin I onBlood Glucose Profile in Diabetic KKAy Mice

In this experiment, the animals in each of the three treatment groups of7 diabetic KKAy mice were orally administered (by gavage) 200, 400, and600 nmole/kg/day des-aspartate-angiotensin I in volume of 0.1 mL water,respectively. Animals in the control group were similarly administered0.1 mL water. The treatment period was 4 weeks. Oral glucose tolerancetest was carried out as described in EXAMPLE 7, except that blood wassampled at 0 and 30 min. The data, presented in Table 5, show thatdes-aspartate-angiotensin I exerts significant hypoglycaemic action atan oral dose of 600 nmole/kg.

Example 10 Effect of Orally-administered Des-aspartate-angiotensin I onBlood Glucose Profile in Diabetic GK Rats

Diabetic GK rats were divided into three groups, consisting of sixanimals in each group. Animals in groups 1 and 2 were administered, bygavage, one of the following: 400 or 600 nmole/kg ofdes-aspartate-angiotensin I in a volume of 0.2 mL water daily for up to8 weeks. Control animals in group 3 were similarly administered 0.2 mLwater. Thirty min oral glucose tolerance test was performed fortnightlyas a spot test to detect significant hypoglycaemic action (if any) thatarose from des-aspartate-angiotensin I treatment. As shown in FIG. 3,significant hypoglycaemic action was detected at 6 weeks of treatment.Animals were treated for a further 2 weeks and a full oral glucosetolerance test was performed thereafter. The data, presented in FIG. 4,show that the hypoglycaemic action of des-aspartate-angiotensin I wasalso significantly expressed at 8 weeks of treatment. The results ofthis study show that des-aspartate-angiotensin I is an effective oralhypoglycaemic agent and can be used to treat the hyperglycemia indiabetes.

Example 11 Effect of Orally-Administered Des-aspartate-angiotensin I onInsulin-induced Translocation of Glucose Transporter-4 (GLU4) to PlasmaMembrane in Skeletal Muscles of Diabetic GK Rats

The diabetic GK rats described in EXAMPLE 9 were similarly treated foranother week. At 9 weeks of treatment, animals were fasted overnight andhalf the animals in each group were intraperitoneally administered withPBS and the other half with insulin (0.5 U/kg). After 30 minutes, theanimals were sacrificed by cervical dislocation. Hind limb skeletalmuscles were rapidly excised and frozen in liquid nitrogen. Musclesamples were stored at −80° C. until used. The hindlimb muscles weresubsequently thawed over an ice pack and plasma membranes were preparedas described by Dombrowaski et al. Thirty μg of protein from the 25%interface of the discontinued sucrose gradient was subjected toSDS-PAGE, transferred to PVDF sheets and incubated with anti-GLUT4polyclonal primary antibody (1:800), washed and followed by incubationwith anti-goat secondary antibody (1:10,000). The bands were detected at49 kDa. The data, presented in FIG. 5, show thatdes-aspartate-angiotensin I significantly enhances the insulin-inducedtranslocation of GLUT4 to the plasma membrane in skeletal muscle ofdiabetic GK rats. As insulin resistance is the hallmark of diabetes, itis concluded that the des-aspartate-angiotensin I exerts markedhypoglycaemic action in diabetic GK rats by overcoming insulinresistance in these animals. This is especially so as serum level ofinsulin was not significantly affected in the same diabetic GK rats thatwere treated with 600 nmole/kg of des-aspartate-angiotensin I (see FIG.6 for details).

Example 12 Kit

The present invention provides a kit comprisingdes-aspartate-angiotensin I, its derivative, a functional part and/or ananalogue thereof for the treatment and/or prophylaxis of at least oneviral infection and/or for the induction of hypoglycaemia and/orreduction of hyperglycaemia. There is also provided a kit for thetreatment and/or prophylaxis of a hyperglycaemia-related conditionexcluding renal-related disorders. The kit may further compriseinformation and/or instruction pertaining to its use.

TABLE 3 Percentage Weight Change of Individual Mouse Post InfectionPeriod Saline treated mice Des-aspartate-angiotensin I treated mice (inDays) 1 2 3 4 5 6 7 1 2 3 4 5 6 7 0 100.0 100.0 100.0 100.0 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 1 99.4 98.1 98.7 99.299.5 99.3 94.8 99.4 100.4 99.0 98.4 99.3 98.5 98.9 2 96.5 97.1 97.5 97.998.2 96.6 87.8 99.3 99.8 96.3 97.1 96.6 97.3 95.8 3 89.8 96.8 96.4 96.895.8 93.9 86.0 99.4 99.0 90.3 94.6 88.6 94.7 93.9 4 88.2 93.2 93.1 86.085.7 87.5 81.5 97.5 96.6 85.7 86.2 83.3 92.1 90.8 5 87.7 86.5 90.9 80.782.0 84.7 80.1 90.3 91.7 82.8 85.7 81.0 90.1 89.0 6 81.8 82.9 86.7 76.878.7 83.4 78.6 87.5 87.4 80.8 82.7 78.2 85.2 88.8 7 80.9 81.1 84.1 69.877.4 78.4 73.9 85.0 83.8 80.7 76.8 73.9 82.8 84.4 8 74.6 79.4 81.5 65.474.9 76.3 69.4 86.2 80.4 79.7 72.9 71.2 83.2 82.4 9 71.8 75.3 80.1 died72.7 74.0 65.1 90.3 78.8 81.4 69.9 71.8 80.5 78.6 10 70.8 70.6 76.4 69.471.0 61.0 93.0 77.5 80.5 69.0 73.2 82.3 77.7 11 69.6 died 72.6 68.2 68.9died 99.3 77.1 82.1 67.7 75.8 88.9 74.5 12 68.8 died died died 101.278.9 80.0 64.9 80.1 88.9 74.5 13 died 80.8 76.6 63.0 82.8 97.4 72.3 1482.6 75.2 61.3 86.3 102.8 74.0 15 84.3 83.0 58.9 90.1 76.7 16 89.4 90.358.9 94.6 78.6 17 94.8 93.5 58.7 99.3 81.2 18 98.7 99.1 died 101.0 83.219 100.9 100.8 86.8 20 91.8 21 95.7 22 98.4 23 100.1

TABLE 4 Dose of des- Serum glucose level at aspartate- differentsampling time (nmole/dL) angiotensin I 0 min 30 min 60 min 120 minControl 8.3 ± 0.6 27.3 ± 1.9  22.2 ± 1.3  12.2 ± 1.3  100 nmole/kg 7.9 ±0.9 21.2 ± 1.6* 13.4 ± 1.3* 9.9 ± 0.5 200 nmole/kg 6.9 ± 0.9 16.0 ± 1.4*14.5 ± 0.9* 8.7 ± 1.2 400 nmole/kg 6.5 ± 0.6 15.4 ± 1.6* 13.0 ± 1.1* 8.8± 0.6 *Significantly different from the corresponding value of theControl (p < 0.05, ANOVA post hoc Tukey test)

TABLE 5 Dose of des- Serum glucose level at different aspartate-sampling time (nmole/dL) angiotensin I 0 min 30 min Control 9.0 ± 1.033.7 ± 1.7 200 nmole/kg 8.9 ± 1.0 37.7 ± 2.6 400 nmole/kg 9.1 ± 1.6 26.0± 3.6 600 nmole/kg 8.9 ± 0.7  17.5 ± 1.9* *Significantly different fromthe corresponding value of the Control (p < 0.05, ANOVA post hoc Tukeytest)

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1. A method of treatment of at least one hyperglycaemia-related condition comprising administering to a subject in need thereof des-aspartate-angiotensin I, wherein the hyperglycaemia-related condition is not a renal-related disorder.
 2. The method according to claim 1, wherein the hyperglycaemia-related condition is type I diabetes, obesity, and/or bulimia nervosa.
 3. The method according to claim 1, wherein the des-aspartate-angiotensin I, is administered in a effective amount.
 4. The method according to claim 1, wherein the des-aspartate-angiotensin I, is administered in combination with at least one pharmaceutically-acceptable carrier, excipient, diluent and/or adjuvant. 